Using a novel PCR-based method (Quantitative Competitive-PCR, QC-PCR), for[unreadable] quantifying HIV-1 RNA we recently discovered unexpectedly high levels of[unreadable] cell-free virus in human plasma. Virus titers determined by this technique[unreadable] were significantly related to disease stage, CD4+ lymphocyte counts, p24[unreadable] antigen levels, and therapy with AZT, and correlated with, but exceeded[unreadable] infectious virus titers in plasma by an average of 60,000-fold (range 8 to[unreadable] 1,200,000; n+66). The biophysical, biological, and genetic basis of this[unreadable] high but variable ratio of total HIV-1 particles to infectious units is[unreadable] completely unknown, as are the contribution of these virus populations to[unreadable] pathogenesis. We will pursue four approaches to rigorously examine the[unreadable] content, composition, biophysical and biological properties of HIV-1[unreadable] virions in plasma, and the clinical correlates of these properties: (1)[unreadable] Using HIV-1 isolated from human plasma, we will determine total virion[unreadable] counts; culturable infectious units; and virion content of (viral) RNA and[unreadable] DNA, structural proteins including gag, pol, env gp 120/gp41, an host cell[unreadable] derived proteins, including MHC Class I, MHC Class II, and beta2-[unreadable] microglobulin, as well as immunoglobulin and complement. We also determine[unreadable] the buoyant density and ultrastructure of virus and/or viral immune[unreadable] complexes in plasma. (2) We will define the biologic and genetic basis for[unreadable] the observed high ratios of total virus to infectious units in plasma by[unreadable] determining the stage(s) in the HIV-1 replication cycle where replication[unreadable] is aborted, and by directly measuring the genetic and functional integrity[unreadable] of tat and RT genes from plasma virus. (3) In patients undergoing rapid[unreadable] changes in viral replication (e.g., in acute infection), and in patients[unreadable] identified in prospective natural history studies as exhibiting rapid or[unreadable] slow disease progression, we will determine if quantitative or qualitative[unreadable] measures of plasma virus content or composition are significantly[unreadable] associated with clinical course and outcome. (4) Using plasma samples from[unreadable] patients treated with novel single agent and combination antiretroviral[unreadable] therapies, we will determine the kinetic profile and magnitude of change in[unreadable] plasma virus as determined by quantitative measurements of viral RNA,[unreadable] infectious units, an p24 antigen. We will also determine the effect of[unreadable] these novel therapies on the biochemical and genetic composition of virus[unreadable] that evolves during and after therapy. The studies proposed will be[unreadable] performed by a collaborative group of investigators each of whom has[unreadable] specialized technical and/or clinical expertise directly related to the[unreadable] aims of this project. The anticipated results are likely to expand, in a[unreadable] new direction, our understanding of basic virologic and host mechanisms[unreadable] underlying HIV-1 pathogenesis, and in addition, should contribute[unreadable] importantly to the development of improved markers for assessing the[unreadable] activity of antiretroviral therapies in vivo.